Interleukin 16 is a potent anti-HIV-1 agent. Previously, we demonstrated that at subnanomolar concentrations the C-terminal 130-amino acids (aa) of IL-16 constitutively secreted by CD4 transfectants renders these cells resistant to HIV-1. We and others also demonstrated that in CD4 T cells, IL-16 mediated HIV inhibition through repression of HIV LTR activity. In this proposal we will focus on three aspects of anti-HIV-1 activity of IL-16. First, we will search for more stable, potent version of IL-16 and molecularly dissect its anti-HIV and chemoattractant activities. We will make gene constructs expressing several modified versions of human IL-16 with a heterologous signal peptide. Stable human CD4 transfectants will be generated. The expression, stability, secretion, and anti-HIV-1 activity of these various versions of IL-16 will be compared. We will also generate several single point mutants of IL-16 to dissect their anti-HIV and chemo attractant activities. Second, we will test the synergy between IL-16 and chemokines in anti-HIV-1 activity. We will generate retroviral gene constructs expressing IL-16 and methionine-modified RANTES or methionine-modified SDF-1beta. The recombinant retroviral particles produced by stable packaging cells will be used to transduce primary human CD4 T cells. The expression of transgenes and the effect of transgene products on both macrophage-tropic and T cell line-tropic HIV-1 replication, CD4, CXCR4 or CCR5 expression, and cell growth will be tested. In parallel, we will make rIL-16, rMet-SDFlbeta, and rMet-RANTES in bacteria. The synergy of these proteins in anti-HIV activity will be quantified. Third, we will develop an ex vivo procedure to regulate IL-16 and chemokine expression in a murine model. Using this model, the long-term regulation and safety of IL-16 and chemokine secretion in vivo will be evaluated. We will generate retroviral constructs co-expressing murine IL-16 and Met-RANTES or Met-SDF-lbeta under a tetracycline-inducible promoter. The inducibility of transgene expression will be tested in genetically manipulated mouse CD4 T cells in vitro. If the results are encouraging, we will transplant these cells into syngeneic mice. The level of transgene expression will be regulated by doxycycline administered in drinking water. TheserumlevelofIL-16andchemokinesandpotentialsignsof inflammation will be closely monitored. We hope these studies will lead to an IL-16- and chemokine-based therapeutic strategy for the treatment of AIDS.